CN101377502A - Tumor-associated antigen 72-4 chemiluminescence immune analysis determination reagent kit and preparing method thereof - Google Patents
Tumor-associated antigen 72-4 chemiluminescence immune analysis determination reagent kit and preparing method thereof Download PDFInfo
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- CN101377502A CN101377502A CNA2007101211688A CN200710121168A CN101377502A CN 101377502 A CN101377502 A CN 101377502A CN A2007101211688 A CNA2007101211688 A CN A2007101211688A CN 200710121168 A CN200710121168 A CN 200710121168A CN 101377502 A CN101377502 A CN 101377502A
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Abstract
The invention relates to the medical field of immunoassay, more specially, the invention provides a tumor associated antigen 72-4 chemiluminescent immunoassay detection kit and a preparation method thereof. The kit of the invention comprises 1) tumor associated antigen 72-4 calibrators, 2) solid-phase vectors which are coated by tumor associated antigen 72-4 monoclonal antibodies, 3) enzyme markers for the tumor associated antigen 72-4 monoclonal antibodies and 4) chemiluminescent substrates which are acted by the enzyme. Further, the preparation method of the kit according to the invention comprises the following steps: 1) preparing the tumor associated antigen 72-4 calibrators, 2) coating the solid-phase vectors with the tumor associated antigen 72-4 monoclonal antibodies, 3) marking the tumor associated antigen 72-4 monoclonal antibodies with the enzyme, 4) packaging the tumor associated antigen 72-4 calibrators, the enzyme markers and the chemiluminescent substrates functioned by the enzyme and 5) assembling finished products. The kit of the invention has the advantages of convenience, rapidness, sensitivity, stability and the like.
Description
Technical field
The present invention relates to the immunoassay medical domain, particularly, the invention provides a kind of tumor-associated antigen 72-4-4 chemiluminescence immune analysis quantitative determination reagent kit and preparation method thereof.
Background technology
The cancer of the stomach case fatality rate occupies first of the various malignant tumours, also is one of China's common malignancy.If can carry out instant examination, will greatly reduce the incidence of cancer of the stomach case fatality rate so to this disease.Tumor-associated antigen 72-4-4 (CA72-4) belongs to the glycoprotein carcinomebryonic antigen, mainly be present in the human adenocarcinoma tissue, be the mark of gastroenteric tumor, in healthy people and benign disease blood samples of patients, seldom occur, and CA72-4 level and tumor development process, tumour size there are good correlativity.Therefore, CA72-4 for the monitoring of the diagnosis of cancer of the stomach, therapeutic process and postoperative curative effect judge significant.
Obtained its monoclonal antibody by the cell membrane component immune mouse of Colcher by breast cancer hepatic metastases cell in 1981, for its immunology detection is opened up new page.Up to now, the detection method of CA72-4 mainly contains: radiommunoassay, enzyme-linked immunosorbent assay and chemiluminescence immune assay etc.Radiommunoassay because of it uses radioelement thing that serves as a mark, therefore has certain contaminative to environment, and exists instrument cost expensive, and sensitivity is not high, complicated operation, measurement result instability, shortcoming such as the reagent holding time is short.Enzyme immunoassay (EIA) sensitivity is low, and influence factor is more, easily causes false negative and false positive.
Chemical luminescence immune analysis reagent box of the prior art is the luminous measuring system of closed full-automatic chemical, needs the expensive luminous measuring instrument of full-automatic chemical, promotes the use of thereby limited, and can't be widely used in clinical diagnosis and research work.The present invention uses the enzymatic luminous substrate on the basis of enzyme-linked immuno assay, by the chromogenic substrate in the light signal replacement enzyme-linked immuno assay that detects the luminous substrate generation, thereby its sensitivity improves greatly, and applicability easy and simple to handle is wide, both can be applicable to the open luminous measuring instrument of semi-automatic chemistry, also can be used for full automatic measuring system, can realize fast detection the in enormous quantities, use cost is low, easier applying.
Summary of the invention
The present inventor proposes and has finished the present invention in order to address the above problem.
The purpose of this invention is to provide a kind of tumor-associated antigen 72-4-4 chemiluminescence immune analysis quantitative determination reagent kit.
A further object of the present invention provides a kind of method for preparing the mentioned reagent box.
Comprise according to tumor-associated antigen 72-4 of the present invention-4 chemiluminescence immune analysis quantitative determination reagent kit:
1) tumor-associated antigen 72-4-4 calibration object;
2) solid phase carrier of tumor-associated antigen 72-4-4 monoclonal antibody bag quilt;
3) tumor-associated antigen 72-4-4 monoclonal antibody enzyme labeling thing; And
4) chemical luminous substrate that above-mentioned enzyme acted on.
According to kit of the present invention, wherein, described solid phase carrier is microwell plate, plastic bead, plastic tube or magnetic-particle.
According to kit of the present invention, wherein, described marker enzyme is alkaline phosphatase or horseradish peroxidase.
According to kit of the present invention, wherein, described chemical luminous substrate is 1,2-two oxidative ethane analog derivatives, luminol or different luminol.
Preferably, described 1 in the mentioned reagent box, 2-two oxidative ethane analog derivatives are (diamantane)-1,2-two oxidative ethanes, 3-(2 '-the spiral diamantane)-4-methoxyl-4-(3 " the phosphorus acyloxy) phenyl-1,2-two oxidative ethanes, CSPD or CDP-Star.
The invention provides a kind of method for preparing the mentioned reagent box, may further comprise the steps:
1) preparation tumor-associated antigen 72-4-4 calibration object;
2) with tumor-associated antigen 72-4-4 monoclonal antibody bag by solid phase carrier;
3) with enzyme labeling tumor-associated antigen 72-4-4 monoclonal antibody;
4) tumor-associated antigen 72-4-4 monoclonal antibody of packing above-mentioned tumor-associated antigen 72-4-4 calibration object, enzyme labeling and chemical luminous substrate that this enzyme is acted on; And
5) be assembled into finished product.
The method according to this invention, preferred, described bag is by the step 2 of solid phase carrier) may further comprise the steps:
I) bag quilt
Adopting 0.05M pH value is that 9.6 carbonate buffer solution or 0.046M pH value are that 4.6 the citrate buffer solution and tumor-associated antigen 72-4-4 monoclonal antibody of debita spissitudo are mixed and made into coating buffer, and it is carried on the solid phase carrier;
II) with physiological saline washing above-mentioned solid phase carrier; And
III) sealing
The preparation confining liquid comprises 0.2g NaH based on the described confining liquid of 1000mL
2PO
42H
2O, 2.9gNaH
2PO
412H
2O, 10g BSA and 1mL biological preservative, the pH value of described confining liquid is 7.0～7.6, then the gained confining liquid is carried on the solid phase carrier after the above-mentioned washing.
Particularly, described method for coating can comprise:
I) bag quilt
The 0.05M pH value that adopts 1000mL is that 9.6 carbonate buffer solution comprises the natrium carbonicum calcinatum of 1.59g and the sodium bicarbonate deionized water solution of 2.94g, or the 0.046M pH value of 1000mL is that 4.6 citrate buffer solution comprises the citric acid of 4.44g and the trisodium citrate deionized water solution of 7.3g is made damping fluid, be mixed and made into coating buffer with tumor-associated antigen 72-4-4 monoclonal antibody of debita spissitudo, and it is carried on the solid phase carrier;
II) with physiological saline washing above-mentioned solid phase carrier; And
III) sealing
The preparation confining liquid comprises 0.2g NaH based on the described confining liquid of 1000mL
2PO
42H
2O, 2.9gNaH
2PO
412H
2O, 10g BSA and 1mL biological preservative, the pH value of described confining liquid is 7.0～7.6, then the gained confining liquid is carried on the solid phase carrier after the above-mentioned washing.
In said method, preferred, described solid phase carrier is microwell plate, plastic bead, plastic tube or magnetic-particle.
In said method, preferred, described enzyme is alkaline phosphatase or horseradish peroxidase.
In said method, preferred, described chemical luminous substrate is 1,2-two oxidative ethane analog derivatives, luminol or different luminol.
In said method, preferred, described 1,2-two oxidative ethane analog derivatives are (diamantane)-1,2-two oxidative ethanes, 3-(2 '-the spiral diamantane)-4-methoxyl-4-(3 " the phosphorus acyloxy) phenyl-1,2-two oxidative ethanes, CSPD or CDP-Star.
When tumor-associated antigen 72-4-4 monoclonal antibody enzyme labeling thing is tumor-associated antigen 72-4-4 monoclonal antibody of horseradish peroxidase-labeled, what adopt is that the sodium periodate method is carried out mark, its concrete principle is: horseradish peroxidase is that a kind of molecular weight is 44,000 glycoprotein, sugar content reaches 18%.The present invention uses sodium periodate oxidation and the irrelevant glycosyl of enzymatic activity, aldehyde radical that generates and the amino on the antibody form Sciff ' s alkali, then add sodium borohydride it is reduced into stable bond, preserve toward adding the glycerine that adds equivalent in the enzymic-labelled antibody at last.
When tumor-associated antigen 72-4-4 monoclonal antibody enzyme labeling thing is tumor-associated antigen 72-4-4 monoclonal antibody of alkali phosphatase enzyme mark, adopt glutaraldehyde method to connect.
Tumor-associated antigen 72-4-4 monoclonal antibody bag is finished by physisorption by solid phase.The inventor has investigated different buffer systems to the physisorption efficient of tumor-associated antigen 72-4-4 monoclonal antibody on solid phase.
The present invention utilizes the enzymatic chemical luminous substrate, generates the intermediate state material.The photon number of launching when getting back to ground state according to middle excited state material is carried out quantitatively.Chemiluminescence enzyme immunoassay has strengthened chemiluminescent intensity by the use of chemiluminescence intensifier, prolongs fluorescent lifetime.The present invention avoids radioelement to pollute simultaneously, has increased significantly than the sensitivity of enzyme-linked immunosorbent assay.
Kit of the present invention has advantages such as easy, quick, sensitive, stable, and its every index all reaches the analytic approach level of similar import reagent box.And, according to detection system of the present invention is open-sky technique, easy to be quick, does not need the expensive luminous measuring instrument of full-automatic chemical, be particularly suitable for vast middle and small hospital and promote the use of, for clinical diagnosis and research work provide a kind of very valuable detection means.According to kit of the present invention, both effectively utilized the chemiluminescence principle, guaranteed the sensitivity that detects again.In addition, this pattern also is convenient to operation and is produced.
Kit of the present invention has been used the enzymatic luminous substrate, by the chromogenic substrate in the light signal replacement enzyme-linked immuno assay that detects the luminous substrate generation, thereby have a specificity equal with enzyme-linked immuno assay, and sensitivity improves greatly, ratio Enzyme Linked Immunoadsorbent Assay sensitivity now improves about 10 times, and the diagnosis that can be cancer of the stomach provides more special, quick, reliable foundation.
Embodiment
The preparation of embodiment 1 tumor-associated antigen 72-4-4 chemiluminescence immune analysis quantitative determination reagent kit
One, enzyme labelled antibody preparation
(1) horseradish peroxidase-labeled tumor-associated antigen 72-4-4 MONOCLONAL ANTIBODIES SPECIFIC FOR
Dissolving 4.4mg HRP is in 1mL distilled water, add 0.4mL sodium periodate (50mmol/L) stirring at room 20min, through the 1mmol/L sodium-acetate buffer, the pH value is that 4.4 dialysis backs add 8mg tumor-associated antigen 72-4-4 monoclonal antibody, stir 2h, use 200mmol/L NaBH at last
4Reduce, after the dialysis of 0.02M PBS damping fluid, add equal-volume glycerine, preserve below-20 ℃.
(2) alkali phosphatase enzyme mark tumor-associated antigen 72-4-4 MONOCLONAL ANTIBODIES SPECIFIC FOR
The CA72-4 monoclonal antibody is fully dialysed to PBS with glutaraldehyde method and alkaline phosphatase coupling, adds equal-volume glycerine, preserves below-20 ℃.
Two, the preparation of tumor-associated antigen 72-4-4 calibration object
With animal blood serum or albumen damping fluid is matrix, and the pure product of adding tumor-associated antigen 72-4-4 are formulated.The ultimate density of preparation is: 0U/mL, 3U/mL, 15U/mL, 50U/mL, 100U/mL.
Three, the preparation of solid-phase coating plate
(1) bag quilt
Adopting 0.05M pH value is that 9.6 carbonate buffer solution or 0.046M pH value are that 4.6 the citrate buffer solution and tumor-associated antigen 72-4-4 monoclonal antibody of debita spissitudo are mixed and made into coating buffer, and it is carried on the solid phase carrier;
Particularly, described method for coating can comprise:
Natrium carbonicum calcinatum 1.59g
Sodium bicarbonate 2.94g
Deionized water 1000mL
Behind the dissolving mixing, adjust pH value to 9.6, add 5.0mg tumor-associated antigen 72-4-4 monoclonal antibody mixing, add then in each hole of microwell plate, every hole 110 μ L, 4 ℃ are spent the night.
Perhaps,
Citric acid 4.44g
Trisodium citrate 7.3g
Deionized water 1000mL
Behind the dissolving mixing, adjust pH value to 4.6, add 5.0mg tumor-associated antigen 72-4-4 monoclonal antibody mixing, add then in each hole of microwell plate, every hole 110 μ L, 4 ℃ are spent the night.
(2) washing: it is inferior to give a baby a bath on the third day after its birth with physiological saline.
(3) sealing
NaH
2PO
4·2H
2O 0.2g
Na
2HPO
4·12H
2O 2.9g
Bovine serum albumin(BSA) 10g
Proclin300 1mL
Distilled water is settled to 1000mL
The mentioned reagent weighing is put into clean container well, add the distilled water constant volume, the dissolving mixing, measuring the pH value is 7.0.Every hole adds confining liquid 300 μ L respectively, and room temperature was placed 3 hours.Get rid of confining liquid, on thieving paper, pat dry.Room temperature removal moisture drying 24 hours.Carry out vacuum sealing bag immediately.Place behind the envelope and checked not have gas leakage in 15 minutes, if there is gas leakage to need envelope again.2～8 ℃ of preservations of labeling postposition.
Four, enzyme mark monoclonal antibody dilution
Tris 12.120g
BSA 5g
Proclin 1mL
Distilled water 1000mL
Five, antibody concentration is selected
The working concentration of tumor-associated antigen 72-4-4 monoclonal antibody of employing square formation method selection tumor-associated antigen 72-4-4 monoclonal antibody horseradish peroxidase or alkali phosphatase enzyme mark is respectively greater than 1:3000 or 1:4000, and tumor-associated antigen 72-4-4 monoclonal coated antibody uses working concentration greater than 1:5000.
Six, chemical luminous substrate liquid
The compound method of the chemical luminous substrate liquid of horseradish peroxidase used in the present invention (HRP):
A liquid: adding Tris and dense HCl in distilled water, to be made into 0.1M pH value be 8.5 Tris-HCl damping fluid.In this damping fluid, add the Luminol of 4.0mg/mL and 0.3mg/mL to iodophenol.
B liquid: in distilled water, add trisodium citrate and citric acid, be mixed with 0.1M pH value and be 4.6 citrate buffer solution, the superoxol of adding 200mg/mL in this solution.
Using method: before using A liquid is mixed the back with B liquid in the 1:1 ratio and use.
The compound method of the chemical luminous substrate liquid of alkaline phosphatase used in the present invention (ALP):
Tris 24g
HCl 15mL
NaCl 160g
KCl 4g
Proclin300 1mL
Distilled water 1000mL
AMPPD 200mL
Seven, lavation buffer solution
Tris 24g
HCl 15mL
NaCl 160g
KCl 4g
Deionized water 1000mL
Adjust pH value to 7.4.
Eight, semi-manufacture and finished product are formed
The packing of above-mentioned steps products obtained therefrom is semi-manufacture.Extract three parts of process specificitys, accuracy, sensitivity and stable assay approvals out and just can be assembled into quantitative determination reagent kit.Be assembled into also need inspect by random samples behind the kit and just can dispatch from the factory after qualified.
To sum up, in research process of the present invention, the present inventor has at first carried out shaker test and Quality Identification to used starting material, then method for coating is studied, select optimal bag and be cushioned liquid and confining liquid, find best concentration conditions, and, made and can make the enzyme labeling thing keep active dilution for a long time.
Embodiment 2～3 preparation tumor-associated antigen 72-4 of the present invention-4 chemiluminescence immune analysis quantitative determination reagent kits
Except that respectively with plastic bead, magnetic-particle as the carrier, all the other all prepare quantitative determination reagent kit of the present invention with the method identical with embodiment 1.
The using method of embodiment 4 kits of the present invention
The concrete operations of the kit of above embodiment 1 preparation are as follows:
1) all detectable of balance and sample are to room temperature;
2) get the lath of expense;
3) every hole, pipe add 50 μ L enzyme labelled antibodies, calibration object/sample to be tested successively, and vibration mixed it in 30 seconds on micro oscillator;
4) room temperature incubation 60min;
5) wash plate with automatic ELIAS microplate washer, the coated antibody-antigen-hrp-antibody complex that is fixed on the solid phase carrier is separated with bond not;
6) every hole/pipe adding volume is the chemical luminous substrate liquid of 100 μ L, and room temperature incubation 5min utilizes chemiluminescence detector to detect in 5～15min;
7) the double-log data fitting mode of use Log (x)-Log (y) is carried out the foundation of typical curve, the experiment with computing measurement result.
The methodology of embodiment 5 kits of the present invention is identified
It is as follows that kit of the present invention can reach the methodology index:
Sensitivity (point that can distinguish with zero-dose) is less than 0.20U/mL;
Standard curve range is 0～100U/mL;
Precision: (N=30) and batch variation are all less than 10% in batch;
Accuracy: the recovery empirical average of a Senior Three concentration serum in hanging down respectively (concentration is 10U/mL, 25U/mL and 65U/mL respectively) is 101%, 112% and 93% (three mean value);
Specificity: with cross reacting rate≤0.01% of its analog;
Stability: each reagent set splits 37 ℃, investigates after 6 days, and each component is still stable.
The elisa kit of embodiment 6 tumor-associated antigen 72-4 of the present invention-4 chemiluminescence immune analysis quantitative determination reagent kits and Hamburg (Germany) diagnostic companies relatively
Take hospital clinical and collect 100 parts of tumour patient serum specimens, 200 parts of normal human serum samples.Use the kit and the Hamburg kit of the embodiment of the invention 1 to carry out blood examination respectively, the statistical experiment result line correlation analysis of going forward side by side, these two kinds of method height correlations (related coefficient is 0.9809).
The clinical trial comparison of kit of the present invention, experimental result sees the following form:
Wherein the CA72-4 mean value of 200 parts of normal person's blood samples is 0.41U/mL, and positive dividing value is decided to be 3.5U/mL.CA72-4 is sensitive indicators of cancer of the stomach colon cancer, positive rate of this law and Hamburg kit approaching, and all within the bibliographical information scope, this is the clinical use value of this kit of susceptible of proof also.
Claims (11)
1, a kind of tumor-associated antigen 72-4-4 chemical luminescence immune assay determination reagent kit is characterized in that, described kit comprises:
1) tumor-associated antigen 72-4-4 calibration object;
2) solid phase carrier of tumor-associated antigen 72-4-4 monoclonal antibody bag quilt;
3) tumor-associated antigen 72-4-4 monoclonal antibody enzyme labeling thing; And
4) chemical luminous substrate that above-mentioned enzyme acted on.
2, kit as claimed in claim 1 is characterized in that, described solid phase carrier is microwell plate, plastic bead, plastic tube or magnetic-particle.
3, kit as claimed in claim 1 is characterized in that, described enzyme is alkaline phosphatase or horseradish peroxidase.
4, kit as claimed in claim 1 is characterized in that, described chemical luminous substrate is 1,2-two oxidative ethane analog derivatives, luminol or different luminol.
5, kit as claimed in claim 4, it is characterized in that described 1,2-two oxidative ethane analog derivatives are (diamantane)-1,2-two oxidative ethanes, 3-(2 '-the spiral diamantane)-4-methoxyl-4-(3 " the phosphorus acyloxy) phenyl-1,2-two oxidative ethanes, CSPD or CDP-Star.
6, a kind of method for preparing the described kit of claim 1 is characterized in that may further comprise the steps:
1) preparation tumor-associated antigen 72-4-4 calibration object;
2) with tumor-associated antigen 72-4-4 monoclonal antibody bag by solid phase carrier;
3) with enzyme labeling tumor-associated antigen 72-4-4 monoclonal antibody;
4) tumor-associated antigen 72-4-4 monoclonal antibody of packing above-mentioned tumor-associated antigen 72-4-4 calibration object, enzyme labeling and chemical luminous substrate that this enzyme is acted on; And
5) be assembled into finished product.
7, method as claimed in claim 6 is characterized in that, described bag is by the step 2 of solid phase carrier) may further comprise the steps:
I) bag quilt
Adopting 0.05M pH value is that 9.6 carbonate buffer solution or 0.046M pH value are that 4.6 the citrate buffer solution and tumor-associated antigen 72-4-4 monoclonal antibody of debita spissitudo are mixed and made into coating buffer, and it is carried on the solid phase carrier;
II) with physiological saline washing above-mentioned solid phase carrier; And
III) sealing
The preparation confining liquid comprises 0.2g NaH based on the described confining liquid of 1000mL
2PO
42H
2O, 2.9gNaH
2PO
412H
2O, 10g BSA and 1mL biological preservative, the pH value of described confining liquid is 7.0～7.6, then the gained confining liquid is carried on the solid phase carrier after the above-mentioned washing.
As claim 6 or 7 described methods, it is characterized in that 8, described solid phase carrier is microwell plate, plastic bead, plastic tube or magnetic-particle.
9, method as claimed in claim 6 is characterized in that, described enzyme is alkaline phosphatase or horseradish peroxidase.
10, method as claimed in claim 6 is characterized in that, described chemical luminous substrate is 1,2-two oxidative ethane analog derivatives, luminol or different luminol.
11, method as claimed in claim 10, it is characterized in that described 1,2-two oxidative ethane analog derivatives are (diamantane)-1,2-two oxidative ethanes, 3-(2 '-the spiral diamantane)-4-methoxyl-4-(3 " the phosphorus acyloxy) phenyl-1,2-two oxidative ethanes, CSPD or CDP-Star.
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| CNA2007101211688A CN101377502A (en) | 2007-08-31 | 2007-08-31 | Tumor-associated antigen 72-4 chemiluminescence immune analysis determination reagent kit and preparing method thereof |
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|---|---|---|---|
| CNA2007101211688A CN101377502A (en) | 2007-08-31 | 2007-08-31 | Tumor-associated antigen 72-4 chemiluminescence immune analysis determination reagent kit and preparing method thereof |
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Cited By (2)
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| CN102890083A (en) * | 2012-09-28 | 2013-01-23 | 辽宁科骏生物有限公司 | Chemiluminescence substrate solution, kit containing same, and detection method applying chemiluminescence substrate solution or kit |
| CN111198271A (en) * | 2018-11-16 | 2020-05-26 | 山东泽济生物科技有限公司 | Chemiluminescent enzyme-linked immunosorbent assay kit for glyceraldehyde-3-phosphate dehydrogenase detection |
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| CN111198271A (en) * | 2018-11-16 | 2020-05-26 | 山东泽济生物科技有限公司 | Chemiluminescent enzyme-linked immunosorbent assay kit for glyceraldehyde-3-phosphate dehydrogenase detection |
| CN111198271B (en) * | 2018-11-16 | 2023-04-28 | 山东泽济生物科技有限公司 | Chemiluminescent enzyme-linked immunosorbent assay kit for detecting glyceraldehyde-3-phosphate dehydrogenase |
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Owner name: BEIJING KEMEI BIOLOGICAL TECHNOLOGY CO., LTD. Free format text: FORMER OWNER: KEMEI DONGYA BIOLOGICAL TECHNOLOGY CO., LTD., BEIJING Effective date: 20111027 |
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Application publication date: 20090304 |